1. Field of the Invention
This invention relates to a method of mosquito larval control. More specifically, the invention relates to larval control by use of a novel spore-forming bacillus and a unique carrier for dispersal of the bacillus. In particular, the invention relates to a unique strain of spore-forming bacillus belonging to the species Bacillus thuringiensis and a unique buoyant colloidal suspension by which the bacillus is carried and dispersed into known habitats of mosquito larvae.
2. Description of the Prior Art
Two major approaches have evolved in the development of effective agents for larval control. One approach is the use of chemical compounds of varying toxicity. The other approach is the development and use of biological microorganisms with high larvicidal activity.
The major shortcoming of the first approach is that new compounds of increased toxicity must be developed or dosage levels of standard compounds increased to counter elevated, adaptive resistance by insect populations to standard insecticides. An unfortunate byproduct of this approach to date is increased toxicity to other lifeforms within the region of application.
Biological microorganisms to date demonstrate a lower or negligible development of resistance in host vectors. Such biological microorganisms may be developed offering improved lethality against host vectors, improved specificity against particular host vectors, and better methods of application without correspondingly lethal effects upon other lifeforms within the region of application.
Technology in the development of bacterial entomotoxins to control mosquito larvae is well known. Some of the entomotoxins employed have been Bacillus sphaericus Neide, Bacillus sphaericus var fusiformis, and a bicrystalliferous strain of Bacillus thuringiensis. In particular, selected strains of Bacillus thuringiensis have been used in Europe and America as major components of microbial insecticides having a toxic effect on general agricultural and forest insect pests. (A. Krieg: Bacillus thuringiensis, Berliner, Berlin, 1961.) To date, however, use of Bacillus thuringiensis against insects directly harmful to humans without concomitant adverse effects has not been employed. The present invention is in part a unique strain of Bacillus thuringiensis, hereinafter referred to as ONR-60A, having high, long-lasting, selectively toxic effects on mosquito larvae with no apparent adverse effects on other lifeforms.
Information gathered from all the above studies indicates that several requirements must be met by any microbial entomotoxin before it can be safely and practically used in the field. These requirements are: (1) effective larval control, e.g., effective dosage levels having 95% lethality when employed (ED.sub.95), should require less than 10.sup.6 cells/cm.sup.2 of larval pond surface area; (2) the toxin should be formulated as a buoyant colloidal suspension such that a toxic concentration will stabilize just below the surface of the larval habitat; (3) larvicidal activity should be retained under conditions of heat and exposure to ultraviolet radiation; and (4) the environmental impact following the use of a selected entomotoxic formulation must not be damaging to a desired ecological balance or otherwise adversely affect other lifeforms within the zone of entomotoxin application.
Regarding criteria (2) listed above, the natural breeding habitat or mosquito larvae is in ponds, lakes, streams, marshes and the like in the shallow littoral zone. Most mosquito larvae must surface for oxygen and as a consequence feed in the region just below the water's surface. Larvicides employed as oil films asphyxiate the larvae as they surface to breath but have the disadvantage that the oil slick is deletorious to much other flora and fauna as well. Further, field studies indicate larvae avoid areas having such oil slicks.
Dust layer and oil film insecticides designed to float on the surface of the water are strongly affected by wind currents, water currents and the like and have serious problems in application and maintenance. Typical of such problems is the drifting and accumulation of such larvicides upon land areas during application or the formation of scum layers on the surface of the water that subsequently concentrate upon land areas.
Larvicidal formulations having non-buoyant characteristics are disadvantageous in that they are largely ineffective if dispersed in water having greater depth than the feeding zone of the larvae. Effectiveness is reduced even more where the non-buoyant formulation is a contact insecticide.
When microbial entomotoxins are employed, the specificity or range of the toxic effect of the particular microorganism is extremely important since it is this characteristic that principally determines its utility and the extent of its use. For example, within the species Bacillus cereus, some variant strains have pathogenic effects limited to a narrow range of insects while other strains have no observed toxic effects at all even though they have essentially identical morphological characteristics. (Ibuki, et al U.S. Pat. No. 3,651,215). As mentioned supra, Bacillus thuringiensis another species of microorganism having great variation of effects by strains within the species, has had little use to date as an entomotoxin against insects directly harmful to humans. Although some strains have been isolated that are effective against mosquito larvae, they have too narrow a range to be of great utility.
Table I illustrates the wide variation of larvicidal activity, or non-activity, of many strains of Bacillus thuringiensis and clearly points out that larvicidal activity is not associated with any single serotype and that the only real characterization or measure of utility and novelty of a microbial entomotoxin is its larvicidal activity. Persons skilled in the art of working with such entomotoxins can routinely cause trivial mutations of a particular entomotoxic agent having no significantly different larvicidal activity, but having some morphological characteristics changed by the trivial mutation. The present invention contemplates such trivial mutations.
TABLE I ______________________________________ Summary of screening of test culture of Bacillus thuringiensis (BA068) for larvicidal activity against Culex tarsalis (K.L.) 1st instar test larvae. ______________________________________ Isolates Demonstrating Larvicidal Activity Original Designation Serotype Code Number ______________________________________ kurstaki H3a,3b* (2536-9693TW) kurstaki " (2819-9763F) aizawai H7* (1850-9762C) tolworthi H9* (NPI-460) sotto H4a,4b (NPI-180-5-2) thuringiensis H1 (NPI-186-104) thuringiensis " (NPI-185-104) thuringiensis " (NPI-201-113) thuringiensis " (NPI-197-105) thuringiensis " (NPI-198-105) thuringiensis " (NPI-199-105) sotto H4a,4b (NPI-194-101) ______________________________________ Isolates Demonstrating No Larvicidal Activity Original Serotype Code Number ______________________________________ kurstaki H3a,3b* (720619A) B. thuringiensis var.? H5a,5b (730130-1) thuringiensis H1* (1840-182C) finitimus H2* (NPI-451) subtoxious H6* (NPI-456) entomocidus H6* (NPI-457) aizawai H7* (NPI-458) morrisoni H8* (NPI-459) darmastadiensis H10* (NPI-461) ______________________________________ *Cultures marked with an asterisk have had recent confirmation. The remainder are based on information available before 1963, and therefore, there is a possibility those classifications are not accurate.